Detection and assay devices and methods of making and using the same

ABSTRACT

An article such as a biomolecular detector or biosensor having a nonfouling surface thereon includes:(a) a substrate having a surface portion; (b) a linking layer on the surface portion; and (c) a polymer layer formed on the linking layer; and (d) a first member of a specific binding pair (e.g., a protein, peptide, antibody, nucleic acid, etc.) bound to the polymer layer. Methods of making and using the articles are also described.

RELATED APPLICATIONS

This application is related to Ashutosh Chilkoti, Non fouling polymericsurface modification and signal amplification method for biomoleculardetection, US Patent Application Pub. No. US 2007/0072220, publishedMarch 29, 2007 (Docket No. 5405-376) (also published as PCT ApplicationNo. WO 2007/035527 on Mar. 29, 2007), the disclosure of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to devices for biomolecular detection.

BACKGROUND OF THE INVENTION

Microarrays are a powerful and established tool in genomics.¹ Incontrast, the development of antibody (Ab) microarrays into anequivalent tool for proteomics has been limited by: (1) the availabilityof high affinity and specificity antibodies for capture and detection ofprotein biomarkers; (2) the susceptibility of proteins to denaturation;and (3) the propensity of Ab's and protein biomarkers to avidly adsorbto surfaces (commonly referred to as the “non-specific adsorption”problem), which can severely limit the ultimate sensitivity of proteinmicroarrays, especially from complex protein mixtures such as plasma andserum.² One of the primary factors (others include the intrinsicaffinity of the capture antibody and the diffusion of target to themicrospot^(2,3)) that controls the limit-of-detection (LOD) of proteinmicroarrays is the adventitious adsorption of proteins (proteinbiomarkers and antibodies used for detection).

SUMMARY OF THE INVENTION

A first aspect of the present invention is an article (preferably abiomolecular detector or biosensor such as a microarray) having anonfouling surface thereon, the article comprising:

(a) a substrate having a surface portion;

(b) a linking layer on the surface portion; and

(c) a polymer layer formed on the linking layer (e.g., by the process ofsurface-initiated polymerization (SIP) of monomeric units thereon).Preferably, each of the monomeric units comprises a monomer (forexample, a vinyl monomer) core group having at least oneprotein-resistant head group coupled thereto, to thereby form a brushmolecule on the surface portion. The brush molecule preferably comprisesa stem formed from the polymerization of the monomer core groups, and aplurality of branches formed from the head group projecting from thestem; and

(d) a first member of a specific binding pair (e.g., a protein, peptide,antibody, nucleic acid, etc.) non-covalently bound to the polymer layer.

A second aspect of the present invention is a method of making anarticle (preferably a biomolecular detector such as a microarray) havinga nonfouling surface thereon, the method comprising: (a) providing asubstrate having a surface portion;

(b) depositing a linking layer on the surface portion; and (c) forming apolymer layer on the linking layer by the process of surface-initiatedpolymerization of monomeric units thereon, with each of the monomericunits comprising a monomer (for example, a vinyl monomer) core grouphaving at least one protein-resistant head group coupled thereto, tothereby form a brush molecule on the surface portion; the brush moleculecomprising a stem formed from the polymerization of the monomer coregroups, and a plurality of branches formed from the hydrophilic headgroup projecting from the stem; and then (d) non-covalently binding amember of a specific binding pair to the polymer layer.

In some embodiments the polymer comprises a homopolymer ofhydroxy-terminated OEGMA. In another embodiment the polymer comprises ofa copolymer of methoxy-terminated OEGMA and hydroxy-terminated OEGMA. Inother embodiments the polymer comprises of vinyl monomer bearing otherhead groups such as hydroxyl (OH), glycerol, or groups known in the artas kosmotropes (see, e.g., Kane et al., infra).

In some embodiments of the invention, the surface portion comprises amaterial selected from the group consisting of metals, metal oxides,semiconductors, polymers, silicon, silicon oxide, and compositesthereof.

In some embodiments of the invention the linking layer is continuous; insome embodiments of the invention the linking layer is patterned. Insome embodiments of the invention the linking layer is a self-assembledmonolayer (SAM). In some embodiments of the invention the linking layercomprises an initiator-terminated silane or an initiator-terminatedalkanethiol. In other embodiments the linking layer comprises of thedeposition of two layers in separate steps. In the first step, analkylsilane or alkanethiol is deposited on a surface such as silicondioxide or glass or gold, and presents a terminal reactive functionalgroup (e.g., amine). In the next step, a bifunctional molecule, whichcomprises a first functional group reactive towards the terminal grouppresented by the first linking layer is reacted with the first linkinglayer deposited in the first step. The second linker molecule contains asecond moiety group that acts as an ATRP or free radical initiator.

In some embodiments of the invention the surface-initiatedpolymerization is carried out by atom transfer radical polymerization(ATRP); in some embodiments of the invention the surface-initiatedpolymerization is carried out by free radical polymerization.

In some embodiments, the article further comprises a protein, peptide,oligonucleotide or peptide nucleic acid non-covalently bound to thepolymer layer. In some embodiments the protein, peptide, oligonucleotideor peptide nucleic acid coupled to the polymer layer or to the surfaceconsists of or consist essentially of a single preselected molecule(this is, one such molecule is coupled to the surface portion via thebrush molecule, to the exclusion of other different molecules). Thepreselected molecule may be a member of a specific binding pair, such asa receptor.

A further aspect of the invention is a method of detecting a secondmember of a specific binding pair in a sample, comprising the steps of:(a) providing a detector as described herein; (b) contacting a sample(e.g., an aqueous sample or biological fluid) suspected of containingthe second member(s) to the detector; and then (c) determining thepresence or absence of binding of the second member to the first member,the presence of binding indicating the presence of the second member inthe sample. The determining step can be carried out by any suitabletechnique, such as by sandwich assay, as discussed further below.

Arrays. In some embodiments of the foregoing methods and devices, usefulfor the detection of multiple different analytes, the first member ofsaid specific binding pair is non-covalently bound to said polymer at adiscrete probe location, and the biomolecular detector furthercomprises: (e) a plurality of additional first members of a specificbinding pairs non-covalently bound to said polymer layer at a pluralityof additional discrete probe locations to thereby form an array thereon.In some embodiments the array has a density of 5 to 10,000 discreteprobe locations per cm² thereon; in some embodiments the array has adensity of 10,000 to 1 million discrete probe locations per cm² thereon;and in some embodiments the array has a density of 1 million to 1billion discrete probe locations per cm² thereon.

An advantage of the foregoing methods is the variety of techniques bywhich the detecting step can be carried out. For example, the detectingstep may be carried out by: (a) ellipsometry; (b) surface plasmonresonance (SPR); (c) localized surface plasmon resonance using noblemetal nanoparticles in solution or on a transparent surface; (d) surfaceacoustic wave (SAW) devices; (e) quartz-crystal microbalance withdissipation (QCM-D) (e) atomic force microscopy, (f) fluorescencespectroscopy or imaging; (g) autoradiography, (h) chemiluminescentimaging; and (i) optical detection of metal nanoparticles either byextinction or scattering. etc.

Still other aspects of the present invention are explained in greaterdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic diagram of an array of the present invention.

FIG. 2. Synthesis of POEGMA brushes on glass via SI-ATRP. Cleaned slideswere functionalized with APTES in step 1, and modified to present anATRP initiator in step 2. Slides were then immersed in a polymerizationsolution in step 3 to synthesize surface tethered brushes of POEGMA.

FIG. 3. (A) Example of signal and background intensities in an arrayused for generation of IL-6 dose response curves (B) Dose responsecurves of OPG in buffer and serum on POEGMA. (C) Dose response curves ofIL-6 in serum on POEGMA and nitrocellulose. In B and C, the Y-axis showsthe average background subtracted fluorescence intensity in printedspots and the X-axis shows analyte concentration in solution. Error barsrepresent one standard deviation.

FIG. 4. Cy-5 labeled goat anti-rabbit IgG (Jackson) printed on POEGMAsubstrates prepared for both covalent and non-covalent attached toproduce arrays.

FIG. 5. Incubation of the arrays of FIG. 4 with Cy5 labeled goatanti-rabbit IgG yielded similar spot intensities for both immobilizationmethods, however, background levels on the activated slides (covalentlycoupled arrays) increased dramatically.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The disclosures of all UnitedStates patents cited herein are incorporated by reference in theirentirety.

1. Definitions.

“SI-ATRP” as used herein means surface initiated atom transfer radicalpolymerization.

“OEGMA” as used herein refers to oligo(ethylene glycol)methylmethacrylate. “Biological fluid” as used herein may be any fluid ofhuman or animal origin, including but not limited to blood, bloodplasma, serum, peritoneal fluid, cerebrospinal fluid, tear, mucus, lymphfluid, semen, saliva, urine, lavage fluid from a wound or bodilyorifice, etc. Biological fluids generally contain a mixture of aplurality of different proteins therein, and typically contain otherconstituents such as other cells and molecules. Biological fluids may bein their natural state or in a modified state by the addition ofingredients such as reagents or removal of one or more naturalconstituents (e.g., blood plasma), but all typically comprise a mixtureof a plurality of different potential analytes, such as a plurality ofdifferent proteins.

“Kosmotrope”, while originally used to denote a solute that stabilized aprotein or membrane, is also used by those skilled in the art, and isused herein, to denote a substituent or “head group” which, whendeposited on a surface, renders that surface protein-resistant. See,e.g., R. Kane, P. Deschatelets and G. Whitesides, Kosmotropes Form theBasis of Protein-Resistant Surfaces, Langmuir 19, 2388-2391 (2003).Numerous kosmotropes are known and examples include but are not limitedto OEGMA. “Polymer” as used herein is intended to encompass any type ofpolymer, including homopolymers, heteropolymers, co-polymers,ter-polymers, etc., and blends, combinations and mixtures thereof.

“Specific binding pair” as used herein refers to two compounds thatspecifically bind to one another, such as (functionally): a receptor anda ligand (such as a drug), an antibody and an antigen, etc.; or(structurally): protein or peptide and protein or peptide; protein orpeptide and nucleic acid; and nucleotide and nucleotide etc. “Nucleicacids” may be any natural or synthetic nucleic acids, including DNA andRNA, and are typically from 10 to 1,000 nucleotides in length. Typicallythe first member of the specific binding pair (or “probe”) is a protein,peptide or nucleic acid that specifically binds to the second member (or“analyte”) to be detected.

“Analyte” as used herein may be any second member of a specific bindingpair, as described above. Typically the analyte is a constituent orfound in a biological fluid as described herein. Examples of suchanalytes include, but are not limited to: thyroid stimulating hormone,glycosylated hemoglobin, parathormone, prostate-specific antigen (psa),ferritin, natriuretic peptide, folic acid, hepatitis b surface antigen,blood lipoproteins, vitamin D, carcinoembryonic antigen, nuclear antigenantibody, testosterone, homocystine, HIV-1 DNA, ck (cpk) gammaglobulin,etc. The analyte can be a “marker” protein, peptide or other moleculespecifically found in patients infected or afflicted with a microbialinfection, examples of which include but are not limited to Anthrax,Avian influenza, Botulism, Buffalopox, Chikungunya, Cholera,Coccidioidomycosis, Creutzfeldt-Jakob disease, Crimean-Congohaemorrhagic fever, Dengue fever, Dengue haemorrhagic fever, Diphtheria,Ebola haemorrhagic fever, Ehec (E. Coli 0157), Encephalitis,Saint-Louis, Enterohaemorrhagic escherischia coli infection Enterovirus,Foodborne disease, Haemorrhagic fever with renal syndrome, Hantaviruspulmonary syndrome, Hepatitis, Influenza, Japanese encephalitis, Lassafever, Legionellosis, Leishmaniasis, Leptospirosis, Listeriosis,Lousebome typhus, Malaria, Marburg haemorrhagic fever, Measles,Meningococcal disease, Monkeypox, Myocarditis Nipah virus, O'Nyong-Nyongfever, Pertussis, Plague, Poliomyelitis, Rabies, Relapsing fever, RiftValley fever, Severe acute respiratory syndrome (SARS), Shigellosis,Smallpox vaccine—accidental exposure, Staphylococcal food intoxication,Tularaemia, Typhoid fever, West Nile fever, Yellow fever, etc. Theanalyte can be a “marker” protein, peptide or other moleculespecifically found in patients infected or afflicted with a fungalinfection, or viral infection. In all of the preceding examples, theanalyte may be derived from the infectious agent itself, namely microbe,fungus or virus, or may be proteins or other biomarkers (such as lipids,carbohydrates or DNA) that are found in greater or lesser abundance inafflicted individuals as compared to healthy individuals.

2. Substrates.

The present invention can be utilized to form surfaces on a variety ofdifferent types of substrates.

In some embodiments, the article is a label-free optical or massdetector (e.g., a surface plasmon resonance energy detector, an opticalwave guide, an ellipsometry detector, etc.) and the surface is a sensingsurface (e.g., a surface portion that would be in contact with abiological fluid). Examples of such articles include but are not limitedto those described in U.S. Pat. Nos. 6,579,721; 6,573,107; 6,570,657;6,423,055; 5,991,048; 5,822,073; 5,815,278; 5,625,455; 5,485,277;5,415,842; 4,844,613; and 4,822,135.

In other embodiments, the article is a biosensor, an assay plate, or thelike. For example, the present invention may be utilized with opticalbiosensors such as described in U.S. Pat. No. 5,313,264 to Ulf et al.,U.S. Pat. No. 5,846,842 to Herron et al., U.S. Pat. No. 5,496,701 toPollard-Knight et al., etc. The present invention may be utilized withpotentiometric or electrochemical biosensors, such as described in U.S.Pat. No. 5,413,690 to Kost, or PCT Application WO98/35232 to Fowlkes andThorp. The present invention may be utilized with a diamond filmbiosensor, such as described in U.S. Pat. No. 5,777,372. Thus, the solidsupport may be organic or inorganic; may be metal (e.g., copper orsilver) or non-metal; may be a polymer or nonpolymer; may be conducting,semiconducting or nonconducting (insulating); may be reflecting ornonreflecting; may be porous or nonporous; etc. For example, the solidsupport may be comprised of polyethylene, polytetrafluoroethylene,polystyrene, polyethylene terephthalate, polycarbonate, gold, silicon,silicon oxide, silicon oxynitride, indium, tantalum oxide, niobiumoxide, titanium, titanium oxide, platinum, iridium, indium tin oxide,diamond or diamond-like film, etc.

The present invention may be utilized with substrates for “chip-based”and “pin-based” combinatorial chemistry techniques. All can be preparedin accordance with known techniques. See. e.g., U.S. Pat. No. 5,445,934to Fodor et al., U.S. Pat. No. 5,288,514 to Ellman, and U.S. Pat. No.5,624,711 to Sundberg et al., the disclosures of which are incorporatedby reference herein in their entirety.

Substrates as described above can be formed of any suitable material,including but not limited to a material selected from the groupconsisting of metals, metal oxides, semiconductors, polymers(particularly organic polymers in any suitable form including woven,nonwoven, molded, extruded, cast, etc.), silicon, silicon oxide, andcomposites thereof.

Polymers used to faun substrates as described herein may be any suitablepolymer, including but not limited to: poly(ethylene) (PE),poly(propylene) (PP), cis and trans isomers of poly(butadiene) (PB), cisand trans isomers of poly(ispoprene), poly(ethylene terephthalate)(PET), polystyrene (PS), polycarbonate (PC), poly(epsilon-caprolactone)(PECL or PCL), poly(methyl methacrylate) (PMMA) and its homologs,poly(methyl acrylate) and its homologs, poly(lactic acid) (PLA),poly(glycolic acid), polyorthoesters, poly(anhydrides), nylon,polyimides, polydimethylsiloxane (PDMS), polybutadiene (PB),polyvinylalcohol (PVA), polyacrylamide and its homologs such aspoly(N-isopropyl acrylamide), fluorinated polyacrylate (PFOA),poly(ethylene-butylene) (PEB), poly(styrene-acrylonitrile) (SAN),polytetrafluoroethylene (PTFE) and its derivatives, polyolefinplastomers, and combinations and copolymers thereof, etc.

If desired or necessary, the substrate may have an additional layer suchas a gold or an oxide layer formed on the relevant surface portion tofacilitate the deposition of the linking layer, as discussed furtherbelow.

3. Linking (or “Anchor”) layers.

Anchor layers used to carry out the present invention are generallyformed from a compound comprising an anchor group coupled (e.g.,covalently coupled) to an initiator (e.g., directly coupled or coupledthrough an intermediate linking group).

The choice of anchor group will depend upon the surface portion on whichthe linking layer is formed, and the choice of initiator will dependupon the particular reaction used to form the brush polymer as discussedin greater detail below.

The anchoring group may be selected to covalently or non-covalentlycouple the compound or linking layer to the surface portion.Non-covalent coupling may be by any suitable secondary interaction,including but not limited to hydrophobic bonding, hydrogen bonding, Vander Waals interactions, ionic bonding, etc.

Examples of substrate materials and corresponding anchoring groupsinclude, for example, gold, silver, copper, cadmium, zinc, palladium,platinum, mercury, lead, iron, chromium, manganese, tungsten, and anyalloys thereof with sulfur-containing functional groups such as thiols,sulfides, disulfides (e.g., —SR or —SSR where R is H or alkyl, typicallylower alkyl, or aryl), and the like; doped or undoped silicon withsilanes and chlorosilanes (e.g., —SiR₂Cl wherein R is H or alkyl,typically lower alkyl, or aryl); metal oxides such as silica, alumina,quartz, glass, and the like with carboxylic acids as anchoring groups;platinum and palladium with nitrites and isonitriles; and copper withhydroxamic acids. Additional suitable functional groups suitable as theanchoring group include benzophenones, acid chlorides, anhydrides,epoxides, sulfonyl groups, phosphoryl groups, hydroxyl groups,phosphonates, phosphonic acids, amino acid groups, amides, and the like.See, e.g., U.S. Pat. No. 6,413,587.

Any suitable initiator may be incorporated into the anchoring group byintroduction of a covalent bond at a location non-critical for theactivity of the initiator. Examples of such initiators include, but arenot limited to, bromoisobutyrate, polymethyl methacrylate-Cl,polystyrene-Cl, AIBN, 2-bromoisobutyrate, chlorobenzene, hexabromomethylbenzene, hexachloromethyl benzene, dibromoxylene, methylbromoproprionate. Additional examples of initiators include thoseinitators described in U.S. Pat. No. 6,413,587 to Hawker (particularlyat columns 10-11 thereof) and those initiators described in U.S. Pat.No. 6,541,580 to Matyjaszewski et al.

As noted above, a linking group or “spacer” may be inserted between theanchoring group and initiator. The linker may be polar, nonpolar,positively charged, negatively charged or uncharged, and may be, forexample, saturated or unsaturated, linear or branched alkylene,aralkylene, alkarylene, or other hydrocarbylene, such as halogenatedhydrocarbylene, particularly fluorinated hydrocarbylene. Preferredlinkers are simply saturated alkylene of 3 to 20 carbon atoms, i.e.,—(CH₂)₄— where n is an integer of 3 to 20 inclusive. See, e.g., U.S.Pat. No. 6,413,587. Another preferred embodiment of the linker is anoligoethyleneglycol of 3 to 20 units, i.e., (CH₂CH₂O)_(n) where n rangesfrom 3 to 20.

The anchoring layer may be deposited by any suitable technique. It maybe deposited as a self-assembled monolayer. It may be created bymodification of the substrate by chemical reaction (see, e.g., U.S. Pat.No. 6,444,254 to Chilkoti et al.) or by reactive plasma etching orcorona discharge treatment. It may be deposited by a plasma depositionprocess. It may be deposited by spin coating or dip coating. It may bedeposited by spray painting. It may also be deposited by deposition,printing, stamping, etc. It may be deposited as a continuous layer or asa discontinuous (e.g., patterned) layer.

In some preferred embodiments, the substrate is glass, silicon oxide orother inorganic or semiconductor material (e.g., silicon oxide, siliconnitride) and compound semiconductors (e.g., gallium arsenide, and indiumgallium arsenide) used for microarray production.

In some preferred embodiments, the anchoring group is a silane orchlorosilane (e.g., -SiR₂Cl wherein R is H or alkyl, typically loweralkyl, or aryl).

In some preferred embodiments, the linking layer comprises of thedeposition of two layers in separate steps. In the first step, ananchoring layer of alkylsilane or alkanethiol is deposited on a surfacesuch as silicon dioxide or glass or gold, and presents a terminalreactive functional group (e.g., amine). In the next step, abifunctional molecule, which comprises a first functional group reactivetowards the terminal group presented by the first linking layer isreacted with the first linking layer deposited in the first step. Thesecond functional group of the bifunctional molecule contains a moietygroup that acts as an ATRP or free radical initator.

4. Brush polymer formation.

The brush polymers are, in general, formed by the polymerization ofmonomeric core groups having a protein-resistant head group coupledthereto. Any suitable core vinyl monomer polymerizable by the processesdiscussed below can be used, including but not limited to styrenes,acrylonitriles, acetates, acrylates, methacrylates, acrylamides,methacrylamides, vinyl alcohols, vinyl acids, and combinations thereof.

Protein resistant groups may be hydrophilic head groups or kosmotropes.Examples include but arc not limited to oligosaccharides, tri(propylsulfoxide), hydroxyl, glycerol, phosphorylcholine, tri(sarcosine)(Sarc), N-acetylpiperazine, betaine, carboxybetaine, sulfobetaine,perrnethylated sorbitol, hexamethylphosphoramide, an intramolecularzwitterion (for example, —CH₂N⁺(CH₃)₂CH₂CH₂CH₂SO₃ ⁻) (ZW), and mannitol.Additional examples of kosmotrope protein resistant head groups include,but are not limited to:

-(EG)₆OH;

—O(Mannitol);

—C(O)N(CH₃)CH₂(CH(OCH₃))₄CH₂OCH₃;

—N(CH₃)₃ ⁺Cl⁻/—SO₃ ⁻Na⁺;

—N(CH₃)₂ ⁺CH₂CH₂SO₃ ,

—C(O)Pip(NAc);

—N(CH₃)₂ ⁺CH₂CO₂ ;

—O([Blc-α(1,4)-Gle-β(1)-]);

—C(O)(N(CH₃)CH₂C(O))₃N(CH₃)₂;

—N(CH₃)₂ ⁺CH₂CH₂CH₂SO₃ ⁻;

—C(O)N(CH₃)CH₂CH₂N(CH₃)P(O)(N(CH₃)₂)₂; and

—(S(O)CH₂CH₂CH₂)₃S(O)CH₃.

See, e.g., R. Kane et al., Langmuir 19, 2388-91 (2003)(Table 1).

A particularly preferred protein resistant head group is poly(ethyleneglycol), or “PEG”, for example PEG consisting of from 3 to 20 monomericunits.

Free radical polymerization of monomers to form brush polymers can becarried out in accordance with known techniques, such as described inU.S. Pat. No. 6,423,465 to Hawker et al.; U.S. Pat. No. 6,413,587 toHawker et al.; U.S. Pat. No. 6,649,138 to Adams et al.; US PatentApplication 2003/0108879 to Klaerner et al.; or variations thereof whichwill be apparent to skilled persons based on the disclosure providedherein.

Atom or transfer radical polymerization of monomers to thin' brushpolymers can be carried out in accordance with known techniques, such asdescribed in U.S. Pat. No. 6,541,580 to Matyjaszewski et al.; U.S. Pat.No. 6,512,060 to Matyjaszewski et al.; or US Patent Application2003/0185741 to Matyjaszewski et al., or variations thereof which willbe apparent to skilled persons based on the disclosure provided herein.

In general, the brush molecules formed by the processes described herein(or other processes either known in the art or which will be apparent tothose skilled in the art based upon the present disclosure), will befrom 2 or 5 up to 100 or 200 nanometers in length, or more, and will bedeposited on the surface portion at a density of from 10, 20 or 40 up to100, 200 or 500 milligrams per meter², or more.

In some preferred embodiments, the polymer layer is formed by SI-ATRP ofOEGMA to form a poly(OEGMA) film. In particularly preferred embodiments,the polymer layer is a functionalized poly(OEGMA) film prepared(preferably in a single step) by copolymerization of a methacrylate andmethoxy terminated OEGMA.

Preparation of substrate and polymer layer far deposition. Prior todeposition of the first member of the specific binding pair, thesubstrate and polymer layer are macroscopically dry or at leastmacroscopically dry (that is, dry to the touch or dry to visualinspection, but retaining bound water or water of hydration in thepolymer layer). To enhance immobilization, it is preferable that thepolymer layer retain bound water or water of hydration (or statedotherwise, that the article includes water consisting of or consistingessentially of waters of hydration, but not bulk surface water). Whenthe substrate with polymer layer has been stored in desiccated form,this can be achieved by quickly hydrating, dipping, or contacting thepolymer layer to water and then blow drying the surface (e.g., with anitrogen or argon jet), or by simply exposing the polymer layer toambient air for a time sufficient for water of hydration to be boundfrom the atmosphere by the polymer layer.

Deposition and post-deposition drying. The first member of the specificbinding pair (as described above) can be deposited on the polymer layerby any suitable technique such as microprinting or microstamping,including piezoelectric or other forms of non-contact printing anddirect contact quill printing.

When an array is being fomied by the deposition of multiple firstbinding pairs, or “probes”, at discrete probe locations on the polymerlayer, probe densities of 1, 3, 5 or 10, up to 100 or 1000 probelocations per cm² can be made. Modern non-contact arrayers can be usedin the deposition step to produce arrays having up to 1,000,000 probelocations per cm². With dip-pen nanolithography, arrays with up to 1billion discrete probe locations per cm² can be made. It will beappreciated that the specific molecular species at each probe locationcan be different from the others, or that some can be the same (e.g., toprovide some redundancy or control), depending upon the particularpurpose of the array.

After deposition of the first member of the specific binding pair, thedevice is optionally but preferably dried, e.g., by mild desiccation,blow drying, lyophilization, or exposure to ambient air at ambienttemperature, for a time sufficient for the article to be macroscopicallydry or at least macroscopically dry as described above. Again, water ofhydration may remain bound by the polymer layer even though the deviceis macroscopically dry. Once the device is macroscopically dry or atleast macroscopically dry, it may be sealed in a container (e.g., suchas an impermeable or semipermeable polymeric container) in which it canbe stored and shipped to the user. Once sealed in the container, thedevice preferably has, in some embodiments, a shelf life of at least 2to 4 months, and preferably up to 6 months or more, when stored at atemperature of 25 ° C. (e.g., without loss of more than 20, 30 or 50percent of binding activity).

5. Uses and applications of articles.

In some embodiments the present invention is utilized by (a) providingan article as described herein; and then (b) contacting the article to abiological fluid or other composition_(;) containing a second member ofthe specific binding pair, wherein the second member of the specificbinding pair binds to the surface portions. Such uses are particularlyappropriate where the article is a sensor or biosensor as described ingreater detail above.

Any suitable “second member” or “analyte” as described above can bedetected. For example, the second member may be a compound found in ormarker for:

-   -   HEP AJB/C/E, Influenza A/B    -   Common and Antibiotic-Resistant Cocci, TB, Syphillis    -   HIV, HCV, HTLV, HPV, Herpes Simplex, Chlamydia, Ghanaian, West        Nile, Chlarnydiazyme    -   CMV, Rubella, TOXO, TPHA, Lyme disease    -   ehrlichiosis, anaplasmosis, bartonellosis, typhus, Q fever,        tickborne spotted fevers, actinomycete

Fungal infection markers:

-   -   aspergillosis, blastomycosis, candidiasis, coccidioidomycosis,        cryptococcosis, histoplasmosis, Pneumocystis carinii

The second member may be one or more of, for example:

-   -   A human cytokine, such as IL-1α, IL-1β, IL-2, IL-4, 1L-5, IL-6,        IL-8, IL-10,IL-12, IL-13, IFN-γand TNFα;    -   A human IR chemokine, such as ENA-78, Eotaxin, GROα, IP-10,        MCP-1, MDC, MIG, MIP-1α, MIP-1β, MPIF-1, RANTES and TARC    -   A human angiogenic factor, such as ANG-2, FGF Basic, HB-EGF,        HGF, KGF, PDGF-BB, THVIP-1, Tpo, VEGF, FGF basic, HGF, PDGF-BB,        VEGF; or

A cardiac marker, such as: Apo A-1, Apo B-100, Fibrinogen, Fibronectinand CRP; Acrp-30, A-SAA, MPO, MMP-2 and MMP-9; AI-1 Active, NT-proBNP,P-Selectin, IL-8, IL-6, OPG, PAPP-A and RANKL; and

-   -   a diabetes and/or obesity marker, such as IGFBP-1, IGFBP-3,        Prolactin, Resistin, CRP, ICAM-1, Acrp-30 and MMP-2; MMP-9,        TNF-RII, VCAM-1 and E-Selectin; Leptin, IL-6, C-peptide and HG.

In one embodiment of the invention, a substrate of the inventioncontains a plurality (e.g., one, two or three, up to 20, 30, or 40 ormore) of different first members that each bind to a different one ofthe foregoing second member/analytes at separate and discrete locationson the substrate polymer layer to form an array or microarray that canbe used to test for a plurality of different analytes in the samebiological fluid sample. The plurality of different first members(selected for the corresponding second members/analytes to which theybind) can be selected and deposited on the array to provide a “panel”test for a particular purpose, such as a human cytokine array; a humanIR chemokine array; a human angiogenic factor array, a cardiac markerarray, a diabetes and/or obesity marker array, a cancer array etc.

Binding of the second member of the specific binding pair (analyte) canbe detected by any suitable technique. In some embodiments the analyteis detected by immunometric assay such as a sandwich assay. In someembodiments of a “sandwich” assay, a third binder, that alsospecifically binds to the second member of the binding pair (the“analyte”), is bound to the analyte, and the binding of that thirdbinder is detected (e.g., by labelling of the third binder with adetectable group such as an enzyme, fluorescent group, or radioactivegroup). Such sandwich assays are well known. Numerous assay formats areknown which can be used or adapted to carry out the present invention.See, e.g., U.S. Pat. Nos. 7,312,041; 7,270,970; 7,267,951; 7,247,500;7,229,775; 7,202,028; 7,195,883; 7,166,469; 7,148,016, etc.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXPERIMENTAL

Herein, we demonstrate that eliminating background adsorption in proteinmicroarrays can decrease the LOD by 100-fold in buffer and serum overthe same protein microarrays printed on a conventional substrate (thatdisplays high binding capacity but significant adventitious adsorption)without need for any other changes to the assay protocol. Notably, theLODs are equivalent for assays performed in either buffer or serumtypically, the LODs for most immunoassays obtained in buffer areseverely compromised when complex protein mixtures such as serum areprobed.⁴

We chose to use a poly(oligo(ethylene glycol) methacrylate) (POEGMA)polymer brush as the microarray substrate because it can be convenientlygrown on glass as a high-density brush that limits protein adsorption.⁵The procedure (SI) used to grow the POEGMA brushes on glass issummarized in FIG. 2. Ellipsometry in air of POEGMA brushes grown onoxidized silicon wafers under identical conditions indicated a POEGMAthickness of 105±2 nm.

A non-contact PerkinElmer Piezorray was used to print Ab microarraysonto POEGMA substrates at room temperature and humidity using coatedslides that had been stored on the benchtop in a closed container for upto two months (substrate storage time had no observed effect on assayperformance). Antibodies for IL-6 and Osteoprotegerin (OPG) (R&DSystems) were printed from 50 gg/mL solutions and allowed tonon-covalently absorb into the 100 nm thick polymer brush. Afterprinting, drying of the spots was facilitated by placing the printedslides under vacuum. This printing and drying process provides stableimmobilization of antibody, as arrayed spots of Cy-5 labeled goatanti-rabbit IgG (Jackson) were still visible after high power sonicationin a 1% Tween-20 solution (SI). An advantage of this approach overchemical activation of the POEGMA brushes and subsequent covalentattachment⁶ is the extreme simplicity of the process, as no slideactivation/deactivation steps are required. Non-covalent immobilizationvia dehydration resulted in equivalent levels of immobilized captureantibody when compared to covalent immobilization via disuccinimidylcarbonate (DSC) and carbonyldiimidazole (CDI) activation procedures(SI). A large increase in background was observed by printing onCDI-activated POEGMA, presumably because of incomplete deactivation ofthe surface after printing (SI), which highlights another importantadvantage of printing directly on the polymer brush as opposed tocovalent coupling. Furthermore, we found that drying the arrays afterprinting does not prevent the recognition of analytes by the captureantibodies, nor does it result in bleeding of the spots upon subsequentexposure to liquids during the interrogation of the arrays (FIG. 3a ).We hypothesize that the 100 nm thick POEGMA brush functions as aquasi-3D hydrogel that retains sufficient interfacial water, even duringmacroscopic drying of the printed arrays, to allow retention of antibodystructure and hence function future experiments will test thishypothesis. After printing the capture antibodies, the arrays werestored in vacuum for eventual use in protein assays. Storage time of theAb arrays from 24 h to two weeks had no obvious effect on arrayactivity. An example of this observation can be seen in FIG. 3b arrayedslides used to produce the two dose response curves were printedsimultaneously, however the assays in buffer preceded the assays inserum by two weeks.

IL-6 and OPG Ab arrays were used to directly probe a dilution series ofanalyte-spiked PBS and serum (assay details below). To compare theperformance of these arrays against a commonly used array material, wealso printed the same Ab arrays on commercially available nitrocellulosemembranes (Whatman), which are used because of their ability to providehigh print densities of the capture antibody and hence high signal.Assays on nitrocellulose substrates were performed according to themanufacturer's suggested protocol.

The fluorescence intensity after scanning and background subtraction fordifferent concentrations of IL-6 as a function of analyte concentrationin serum are shown in FIG. 2C for nitrocellulose and POEGMA. The dataclearly show that the fluorescence signal from the printed capture Abspots on nitrocellulose are only visible to a concentration of 10 pg/ml,while the signal on POEGMA is clearly visible down to a concentration of100 fg/ml. Furthermore, despite the incubation and rinse steps, therewas no bleeding of the spots (FIG. 3A), which confirmed the stableimmobilization of the capture antibody. The image in FIG. 3A also showsthat the POEGMA matrix retains its ability to resist non-specificprotein adsorption throughout the entire array fabrication and assayprocess fluorescence intensities of the background areas surroundingprinted spots measured prior to the assay show no increase in intensityupon completion of the procedure (the only background fluorescencedetected on the POEGMA substrates is due to the autofluoresence of theglass slide). This elimination of background signal allows the POEGMAsubstrates to achieve LODs (signal was considered significant if greaterthan three standard deviations above the average of the same Ab spotsexposed to non-spiked serum) that are up to two orders of magnitude moresensitive when compared to traditional nitrocellulose substrates, asshown by the dose-response curves in FIG. 3C.

The POEGMA substrates also provide an improved dynamic range and canquantify protein concentration across six orders of magnitude, as seenin the dose response curves in FIG. 2 and summarized in Table 1. OPGdose response curves in buffer and serum are shown in FIG. 2B toillustrate the important point that the Ab arrays on POEGMA havevirtually identical LODs in buffer and serum. This is in contrast tomost other fluorescence immunoassays, where the LOD is typically ordersof magnitude greater in complex physiological solutions containing highconcentrations of extraneous proteins when compared to LODs determinedin buffer.

TABLE 1 Limits of Detection (LOD) and Dynamic Ranges of Serum-BasedMicroarray Assays on POEGMA Analyte LOD Dynamic Range IL-6 100 fg/mL 100fg/mL-10 ng/mL IL-β 100 fg/mL 100 fg/mL-10 ng/mL TNF-α 100 fg/mL 100fg/mL-10 ng/mL IL-8 100 fg/mL 100 fg/mL-10 ng/mL OPG 1 pg/mL  1 pg/mL-10ng/mL

In conclusion, we have demonstrated antibody arrays on POEGMA brusheswith several significant features: first, the direct physical printingof the capture Abs provides a simple and robust procedure for the stableimmobilization of the capture Abs that avoids the need for chemicalactivation and deactivation of the surface.

Second, the printed microarrays have a practical shelf-life of at leastseveral weeks with no loss in performance. Third, antibody arraysprinted on the POEGMA brushes require during interrogation of the array,which simplifies the assay. Finally, the resistance of the POEGMAbrushes to protein adsorption from solution eliminates background noisein the microarrays stemming from adventitious protein adsorption andleads to LODs as low as 100 fg/mL in serum (which corresponds to 4 fMfor IL-6). The femtomolar LODs in serum and the wide dynamic rangesuggest that these microarrays will he useful for the quantification oflow abundance protein biomarkers directly from complex mixtures withminimal sample pre-processing.

Methods:

Synthesis of POEGMA surfaces: The POEGMA brushes were fabricated onglass as follows (FIG. 2, all chemicals purchased from Sigma): first,glass slides (VWR) were cleaned in a solution of 3:1 H-)SO₄:H₂O₂ for 30minutes. After rinsing with deionized H₂O and drying, the cleaned slideswere immersed in 10% aminopropyltriethoxysilane (APTES) in ethanol for30 min and were then rinsed with ethanol and dried at 120° C. for 3 h(step 1). Slides were then immersed in a solution of 1% bromoisobutyrylbromide and 1% triethylamine in dichloromethane for 30 min, rinsed withdichloromethane and ethanol, and blown dry with N₂ (step 2). Slides werethen immersed for 12 h in a degassed polymerization solution of 5 mg/mLCu(I)Br, 12mg/mL bipyridine and 300mg/mL oligo(ethylene glycol)methacrylate under argon (step 3). Finally, slides were rinsed withdeionized H₂O and blown dry with N₂.

Antibody Immobilization: Cy-5 labeled goat anti-rabbit IgG (Jackson) wasprinted on POEGMA substrates to produce arrays seen in FIG. 4. Thearrays were dehydrated for 24 hours to promote immobilization and thensubjected to high power sonication for 10 minutes in a 1% Tween-20 PBSsolution.

Covalent vs. non-covalent attachment: Identical arrays of mouseanti-goat IgG were printed onto unmodified POEGMA substrates as well assubstrates that had been activated with either disuccinimidyl carbamate(DSC) or carbonyldiimidazole (CDI). Subsequent incubation with Cy5labeled goat anti-rabbit IgG yielded similar spot intensities for bothimmobilization methods, however, background levels on the activatedslides increased dramatically, as shown in FIG. 5. We suggest that theuse of ethanolamine to deactivate unused conjugation sites, and thesubsequent incorporation of large numbers of amide bonds into the POEGMAlayer, as well as residual reactive groups, led to the increase innon-specific protein adsorption and coupling in subsequent steps.

CDI activation protocol: Slides were immersed in a 0.5M solution of CDIin dry dioxane for two hours at 37° C. with stirring and then rinsedwith dry dioxane, dried, and used immediately for printing.

DSC activation protocol: Slides were immersed in a solution of 0.6M DSCand 0.6M 4-(dimethylamino)pyridine in dry acetone for 6 hours withstirring and then rinsed with dry acetone, dried, and used immediatelyfor printing.

Deactivation protocol: Printed slides were immersed in a 0.1M Na Boratebuffer at pH 8.5 for 1 hour, and were then transferred to a 0.1M NaBorate buffer at pH 8.5 with 1M ethanolamine for 1 hour.

Multiplexed sandwich immunoassay details: Arrays were first incubatedwith a dilution series of 100 ml of analyte-spiked PBS or serum for 2 hwith stirring, followed by 100 ml 1 mg/ml biotinylated secondaryantibody in PBS with 1% (w/v) BSA for 1 h. Finally, the arrays weredeveloped by incubation in 100 ml of 1 ug⁻/m1 streptavidin-Cy5 for 30min, and then scanned with an Axon Genepix 4200 fluorescence microarrayscanner. After each incubation step, arrays were washed twice for 30 swith 1% BSA (w/v) and 0.1% (w/v) Tween-20 in PBS.

REFERENCES

1a) Schena et al, 1995 Science 270: 467-470 b) Mantripragada K et al,2004 TIG 20: 87-94 c) Stoughton R B 2005 Annu Rev Biochem 74: 53-82

2a) Angenendt, P., Lehrach, H., Kreutzberger, J., et al. 2005 Proteomics5, 420-425 h) Haab, B. B. 2003 Proteomics 3, 2116-2122. c) Kingsmore, S.F. 2006 Nature Rev Drug Discov 5, 310-320. d) Kusnezow, W., Hoheisel, J.D. 2003 J. Mol. Recognit 16: 165-176 e) Macbeath, G. 2002 NatureGenetics 32, 526-532 d) Pavlickova, P., Schneider, E. M., and Hug, H.2004 Clin Chim Acta 343, 17-35. e) Wilson, D. S., and Nock, S. 2003Angew Chem 42, 494-500. f) Wingren, C., and Borrebaeck, C. A. K. 2004Expert Rev Proteomics 1, 355-364. g) Wingren, C., and Borrebaeck, C. A.K. 2006 Omics 10, 411-427 h) Zhu, H., Bilgin, M., and Snyder, M. 2003Annu Rev Biochem 72, 783-812.

3a) Kusnezow, W., Syagailo, Y. V., Ruffer, S., Klenin, K., Sebald, W.,Hoheisel, J. D., Gauer, C., and Goychuk, I. 2006 Proteomics 6, 794-803b) Kusnezow, W., Syagailo, Y. V., Goychuk, I., Hoheisel, J. D., andWild, D. G. 2006 Expert Rev. Mol. Diagn 6, 111-124

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5a) Ma H W, Li D J, Sheng X, et al. 2006 Langmuir 22: 3751-3756 b) BrownA A, Khan N S, Steinbock L, et al. 2005 Eur. Polym. J. 41: 1757-1765 c)Tugulu, S., Klok, H A 2008 Biomacromolecules, 9: 906-912

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The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-35. (canceled)
 36. A method of detecting the presence of an analyte ina sample suspected of containing the analyte, the method comprising (a)contacting the sample suspected of containing the analyte to abiomolecular detector, wherein the biomolecular detector comprises: (i)a substrate having a surface portion, (ii) a linking layer on thesurface portion, (iii) a polymer layer formed on the linking layer,wherein the polymer comprises monomeric units, with each of themonomeric units comprising a monomer core group having at least oneprotein-resistant head group coupled thereto, to thereby form a brushmolecule on the surface portion; wherein the brush molecule comprises astem formed from the polymerization of the monomer core groups, and aplurality of branches formed from the head group projecting from thestem, and (iv) a probe directly non-covalently bound to the polymerlayer; (b) incubating the sample on surface portion of the biomoleculardetector to permit specific binding between the probe and the analyte,and (c) detecting the binding of the analyte to the probe, whereindetection of the binding of the analyte to the probe indicates thepresence of the analyte in the sample.
 37. The method of claim 36,wherein the probe is a protein, peptide, or nucleic acid.
 38. The methodof claim 36, wherein the probe is an antibody.
 39. The method of claim36, wherein the surface portion of the biomolecular detector comprises amaterial selected from the group consisting of metals, metal oxides,semiconductors, polymers, silicon, silicon dioxide, and compositesthereof.
 40. The method of claim 36, wherein the surface portion of thebiomolecular detector comprises silicon dioxide or gold.
 41. The methodof claim 36, wherein the linking layer of the biomolecular detector iscontinuous.
 42. The method of claim 36, wherein the linking layer of thebiomolecular detector is patterned.
 43. The method of claim 36, whereinthe linking layer of the biomolecular detector comprises a silane layeror a self-assembled monolayer.
 44. The method of claim 36, wherein thebrush molecule of the biomolecular detector is from 5 to 200 nanometersin length.
 45. The method of claim 36, wherein the brush molecule of thebiomolecular detector occurs on the surface portion of the biomoleculardetector at a density from 10 to 500 milligrams per meter².
 46. Themethod of claim 36, wherein the probe is bonded to the polymer layer ofthe biomolecular detector at a density of from 1 milligram per meter² to50 grams per meter².
 47. The method of claim 36, wherein the analyte ispresent in the sample at a concentration of from 0.1 to 100 femtomolesper liter.
 48. The method of claim 36, wherein the detecting comprisesan immunoassay.
 49. The method of claim 36, wherein the sample comprisesa biological fluid.
 50. The method of claim 49, wherein the biologicalfluid comprises blood, blood plasma, serum, peritoneal fluid,cerebrospinal fluid, tear, mucus, lymph fluid, semen, saliva, urine, andlavage fluid from a wound or bodily orifice.